Structure-activity relationships in the oxidation of para-substituted benzylamine analogues by recombinant human liver monoamine oxidase A
Structure-activity relationships in the oxidation of para-substituted benzylamine analogues by recombinant human liver monoamine oxidase A
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DOI：
10.1021/bi990920y
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发表时间：
1999-10-12
期刊：
影响因子：
2.9
通讯作者：
Edmondson, DE
中科院分区：
文献类型：
--
作者：
Miller, JR;Edmondson, DE
单胺氧化酶A（Monoamine oxidase A，MAOA）在胺类神经递质的氧化过程中起着重要作用。为了研究该酶的结构和作用机制，从酿酒酵母中表达并纯化了重组人肝脏MAO A。厌氧滴定的酶只需要1摩尔底物每摩尔酶结合的黄素完全还原。这表明只有一个氧化还原活性基团（即，共价FAD辅因子）参与催化。通过稳态和停流动力学实验测定了17种对位取代苄胺类似物与纯化的MAO A的反应速率和结合亲和力。对于测试的每种底物类似物，稳态转换率（k（cat））和厌氧黄素还原率（k（red））的值相似。对于所测试的每种底物类似物，氘动力学同位素对k（cat）、k（red）、k（cat）/K-m和k（red）/K-s与α，α-[H-2]苄胺的影响相似，范围为6至13，表明α-C-H键断裂在催化中是限速的。从还原半反应实验以及从稳态动力学同位素效应数据测定底物类似物解离常数[Klinman，J. P.，和马修斯，R. G.等人（1985）J. Am. 107，1058-1060]的结果非常一致。解离常数的定量构效关系（QSAR）分析表明，对位取代苄胺类似物与MAO A的结合与取代基的货车德瓦尔斯体积相关性最好，取代基越大结合越紧密。对位取代的苄胺类似物氧化和/或底物类似物依赖性黄素还原的速率与取代基电子效应（sigma）最相关。分离的电子取代基参数（西格玛）场诱导和共振效应提供了一个更全面的治疗的电子相关性。速率与σ的正相关性（ρ类似于2.0）表明在苄基碳位置处发生负电荷发展，并支持质子提取作为α-C-H键裂解的模式。根据提出的几种MAO催化机制和先前发表的牛肝MAO B的结构-活性研究[步行者，M. C.的方法，和Edmondson，D. E.（1994）Biochemistry 33，7088-7098]。
Monoamine oxidase A (MAO A) plays a central role in the oxidation of amine neurotransmitters. To investigate the structure and mechanism of this enzyme, recombinant human liver MAO A was expressed and purified from Saccharomyces cerevisiae. Anaerobic titrations of the enzyme require only 1 mol of substrate per mole of enzyme-bound flavin for complete reduction. This demonstrates that only one redox-active group (i.e., the covalent FAD cofactor) is involved in catalysis. The reaction rates and binding affinities of 17 para-substituted benzylamine analogues with purified MAO A were determined by steady state and stopped flow kinetic experiments. For each substrate analogue that was tested, the rates of steady state turnover (k(cat)) and anaerobic flavin reduction (k(red)) are Similar in value. Deuterium kinetic isotope effects on k(cat), k(red), k(cat)/K-m, and k(red)/K-s with alpha,alpha-[H-2]benzylamines are similar for each substrate analogue that was tested and range in value from 6 to 13, indicating that alpha-C-H bond cleavage is rate-limiting in catalysis. Substrate analogue dissociation constants determined from reductive half-reaction experiments as well as from steady state kinetic isotope effect data [Klinman, J. P., and Matthews, R. G. (1985) J. Am. Chem. Soc. 107, 1058-1060] are in excellent agreement. Quantitative structure-activity relationship (QSAR) analysis of dissociation constants shows that the binding of para-substituted benzylamine analogues to MAO A is best correlated with the van der Waals volume of the substituent, with larger substituents binding most tightly. The rate of para-substituted benzylamine analogue oxidation and/or substrate analogue-dependent flavin reduction is best correlated with substituent electronic effects (sigma). Separation of the electronic substituent parameter (sigma) into field-inductive and resonance effects provides a more comprehensive treatment of the electronic correlations. The positive correlation of rate with sigma (rho similar to 2.0) suggests negative charge development at the benzyl carbon position occurs and supports proton abstraction as the mode of alpha-C-H bond cleavage. These results are discussed in terms of several mechanisms proposed for MAO catalysis and with previous structure-activity studies published with bovine liver MAO B [Walker, M. C., and Edmondson, D. E. (1994) Biochemistry 33, 7088-7098].